This project will develop and apply methods to analyze functioning of a microbicide product vaginal epithelial coating layer - to inhibit transport of HIV virions from semen to tissue and enable microbicide molecules to disable virus. We will contribute two missing capabilities to the microbicide pipeline: (1) mechanistic understanding of interactions between coating properties and anti-viral agents in neutralizing semen-born HIV;and (2) practical experimental and mathematical tools to help design and compare products, prior to animal and human studies. The new methodology combines two complementary procedures. First (Specific Aim 1) we will develop a new bioassay that places a layer of HIV-laden semen (or semen simulant) over a biologically relevant thin microbicide gel layer that rests on a tissue explant (or surrogate) substrate. This configuration simulates in vivo conditions in the vagina that, we hypothesize, play a critical role in microbicide efficacy;the configuration is not achieved in the few current bioassays of microbicide actives within their gel vehicles. Transport of HIV (total virus and infectious virus) to the substrate will be determined - as a direct measure of efficacy of the microbicide product layer. This assay also enables measurement of release kinetics of antiviral agents from the gels, in a more realistic configuration than current methods. Second (Specific Aim 2) we will develop a technique to determine diffusion coefficients within microbicide gels of HIV and active ingredients over the length scales of coating thickness (100 - 500 5m) that have been measured in the human vagina. These coefficients are paramount in governing the time required for virions to migrate from semen to tissue. They provide information about how structural characteristics of a gel coating can impact virion and active ingredient mobilities. Measured coefficients will be input to a mechanistic mathematical model of HIV transport from semen to epithelium, and of its neutralization by co- diffusing antiviral agents from a coating layer, e.g. viral envelope entry inhibitors. The model provides an objective means to distinguish the relative roles of the active ingredient (concentration, potency, diffusion coefficient) and coating layer (thickness, HIV diffusion coefficient), together with viral load in semen, in microbicide product efficacy. The model can help interpret differences between products predicting how parameters of active ingredients and delivery vehicles affect efficacy, and leading to design of better products. Specific Aims 1 and 2 are synergistic, providing new insights about the hypothesis that epithelial coating - initial and sustained - is important to semi-solid microbicide product efficacy. They will contribute unique pharmacokinetic and pharmacodynamic analysis tools to the microbicide pipeline. PUBLIC HEALTH RELEVANCE: This project will create novel, unprecedented methodology to understand and test how microbicide gel products prevent semen-borne HIV from initiating transmission. This methodology will fill a gap in the microbicide pipeline, which is seeking to provide women with products that can prevent infection by HIV and other sexually transmitted pathogens.